UNIVERSIDADE DE LISBOA Faculdade de Medicina Veterinária
L-MESITRAN® IN THE MANAGEMENT OF CANINE OTITIS EXTERNA – A PILOT STUDY EMI MARUHASHI 2015 Lisboa ORIENTADORA Doutora Ana Mafalda Gonçalves Xavier Félix Lourenço
CO-ORIENTADORA Doutora Berta Maria Fernandes Ferreira São Braz
CONSTITUIÇÃO DO JÚRI
Doutor José Henrique Duarte Correia Doutor Luís Miguel Alves Carreira
Doutora Ana Mafalda Gonçalves Xavier Félix Lourenço
UNIVERSIDADE DE LISBOA Faculdade de Medicina Veterinária
L-MESITRAN® IN THE MANAGEMENT OF CANINE OTITIS EXTERNA – A PILOT STUDY
EMI MARUHASHI
DISSERTAÇÃO DE MESTRADO INTEGRADO EM MEDICINA VETERINÁRIA
2015 Lisboa
ORIENTADOR Doutora Ana Mafalda Gonçalves Xavier Félix Lourenço
CO-ORIENTADOR Doutora Berta Maria Fernandes Ferreira São Braz
CONSTITUIÇÃO DO JÚRI
Doutor José Henrique Duarte Correia Doutor Luís Miguel Alves Carreira
Doutora Ana Mafalda Gonçalves Xavier Félix Lourenço
ACKNOWLEDGEMENTS
First and foremost, thank you to my thesis coordinator and mentor, Professor Ana Mafalda. It has been a long road and I am grateful for all the support and belief which you instilled in me. Today I am above all, happy to have gained a friend for life.
Thank you to the orientation of Professor Berta São Braz, without whose dedication and support this thesis could not have been orchestrated in the manner that it was.
To the dermatology dream team – Thank you Prof. Mafalda, Lúcia and Joana, for the time and care dedicated to helping me with the patients in this study, for making hard work fun and for all the laughs and 5-minute “lunch” breaks by the vending machine on 12-hour days filled with overbookings.
To the colleagues at the FMV Hospital - thank you for taking the time to help in recruiting the patients for this study.
To Professor Telmo Nunes – Thank you for having all the patience in the world to teach me the language of statistics and for being a life coach with words of wisdom and honesty. To the owners of the dogs included in this study – thank you for giving me a chance.
To Professor Maria Constança Pomba and her laboratory for establishing the invaluable link between clinical and laboratory results, in particular to Eng. Adriana Belas – Thank you for undertaking the essential work in this project.
To Dr. Isabel Pimenta (Biolotus®) – You believed in this project when it was just a seed waiting to be planted and there was nothing yet to show. Thank you.
To João Miguéns (Biolotus®) – Thank you for the support and hard work in providing material.
To Mr. Joost Postmes (Triticum®) – Thank you for giving us this opportunity and for allowing us to spread the word regarding honey. One step at a time, the world will get there. To Mariana – Your last minute help was invaluable and I know you know I couldn´t have done it without you! Thank you!
To Cristina Tinkerbell – Thank you for your friendship, advice and support. Knowing I could count on you along this journey made the hard times easier to bear.
To Carlita – Your good disposition always brought a smile to my face and made cloudy days brighter.
Mom and Dad – I was never easy but you didn´t give up on me and words are not nearly enough to express my gratitude towards all the love you gave and the sacrifices you made for me to be here. I´m glad I have the rest of my life to keep thanking you. If one day I can be even half the parents you were to me, I will be content.
Jin – The little brother I´ve always looked up to. Thank you for putting up with me for all these years even though you never had a choice. I hope I´ve made you proud.
Bu – The definition of unconditional love. You got me through everything. Thank you. To those of you who go unnamed - Thank you for coming into my life. May you always stay.
Thank you to Triticum® (Maastricht, Netherlands) and Biolotus® (Lisbon, Portugal) for kindly providing the materials and funding necessary for this study.
ABSTRACT
“L-MESITRAN® IN THE MANAGEMENT OF CANINE OTITIS EXTERNA – A PILOT STUDY”
At a time when antimicrobial resistance is rising steadily and the involved microorganisms are demonstrating zoonotic potential, honey and its derived products may prove useful in this ongoing battle. Otitis externa in dogs is considered to be one of the most prominent causes for presentation at veterinary practice. Some of the regularly administered agents in otitis treatments are no longer effective, as resistance has increased, perhaps due to the often long-term periods which are necessary for resolution and the accompanying tendency towards chronicity. In order to address the need for efficient alternative treatments, L-Mesitran® Soft, a medical grade honey gel was used to treat 15 dogs with otitis externa of bacterial and/or fungal involvement. Success was based on clinical score decrease, cytology and owner input over time and with basis on culture results. 70% of enrolled dogs achieved clinical cure between days 7 to 14 and over 90% on day 21, the maximum established time limit, with a confidence interval of 95%. Furthermore, by day 7, 20% of dogs had obtained both clinical and cytological cures. This study was successfully able to demonstrate that the use of L-Mesitran® was effective in managing otitis externa in dogs, including cases in which highly resistant pathogens were present, such as methicillin-resistant Staphylococcus pseudintermedius (MRSP), thus paving the way to future studies.
Key-words: honey; antimicrobial resistance; canine; otitis externa; L-Mesitran® Soft; methicillin-resistant Staphylococcus pseudintermedius (MRSP).
RESUMO
“L-MESITRAN® NO MANEIO DE OTITE EXTERNA CANINA – UM ESTUDO PILOTO”
Numa altura em que nos deparamos com um aumento de bactérias com resistências aos antibióticos e em que os organismos envolvidos apresentam por vezes inclusivamente potencial zoonótico, o recurso ao mel e seus derivados pode ser uma inestimável ferramenta no decurso desta batalha. A otite externa em cães é um estímulo iatrotrópico frequente e das principais causas de idas ao médico veterinário. Alguns dos tratamentos habitualmente utilizados deixaram de ser eficazes à medida que as resistências surgiram, talvez consequência de terapêuticas prolongadas e recorrentes. Neste estudo avaliou-se uma potencial alternativa à terapêutica, recorrendo-se ao L-Mesitran® Soft, uma pomada contendo mel de grau clínico, para tratar 15 cães com otite externa de envolvimento bacteriano e/ou fúngico. A resposta foi considerada positiva de acordo com a diminuição da pontuação clínica, citologia e opinião dos donos, no decorrer do tempo. Estabelecido o limite de 21 dias, 70% dos cães tratados obteve cura clínica entre os dias 7 e 14 e mais de 90% no dia 21, com um intervalo de confiança de 95%. Ainda, até ao dia 7, 20% dos cães havia obtido cura clínica e citológica. Este estudo demonstrou que o L-Mesitran® foi eficaz no maneio dos casos de otite externa, incluindo aqueles em que estavam presentes bactérias com várias resistências aos antibióticos, como é o caso do Staphylococcus pseudintermedius com resistência à meticilina (SPRM), abrindo assim caminho para futuros estudos.
Palavras-chave: mel; resistência antimicrobiana; canina; otite externa; L-Mesitran® Soft; Staphylococcus pseudintermedius resistente à meticilina (SPRM).
GENERAL INDEX
ABSTRACT ... iii
RESUMO ... iv
GENERAL INDEX ... v
LIST OF FIGURES ... viii
LIST OF TABLES ... ix
LIST OF ABBREVIATIONS ... x
I. INTRODUCTION ... 1
1. REVIEW OF CURRICULAR INTERNSHIP – WEST CROSS VETERINARY HOSPITAL, JAPAN ... 1
2. REVIEW OF CURRICULAR INTERNSHIP – FACULTY OF VETERINARY MEDICINE, UNIVERSITY OF LISBON, PORTUGAL ... 2
II. REVIEW OF LITERATURE ... 4
1. HONEY ... 4
1.1. Honey throughout history ... 4
1.2. Honey production ... 4
1.3 General properties of honey ... 5
1.3.1. Enzymes of honey ... 6
1.3.2. Other components of honey ... 7
1.4. Healing properties of honey ... 7
1.4.1. Sugar content ... 7
1.4.2. pH of honey ... 8
1.4.3. Hydrogen Peroxide ... 9
1.4.4. Effect on Biofilms ... 10
1.4.5. Effect on Colonization ... 11
1.4.6. Effect on angiogenesis ... 12
2. ALL HONEYS ARE NOT THE SAME ... 13
2.1. Honey use in medicine ... 13
2.2. Manuka honey ... 14
2.3. Medical grade honey ... 15
2.3.1. Variation among medical grade honeys ... 15
2.3.2. Risks & gamma irradiation ... 16
2.4. Honey as a medical device ... 17
2.5. Generalities in wound healing ... 18
2.5.1. Physical barrier ... 18 2.5.2. Wound acidification ... 18 2.5.3. Debriding action ... 19 2.5.4. Deodorizing effect ... 19 2.5.5. Anti-‐inflammatory effect ... 19 2.5.6. Antitumor effect ... 21 2.5.7. Immuno-‐stimulatory effect ... 21 2.5.8. Antioxidant activity ... 21
2.5.9. Honey versus Silver ... 22
3.1. Triticum® ... 23
3.1.1. Characteristics ... 23
3.1.2. Applications – Human medicine ... 23
3.1.3. Applications – Veterinary medicine ... 25
4. OTITIS ... 26
4.1. General prevalence ... 26
5. OTITIS EXTERNA ... 27
5.1. External ear anatomy ... 27
5.2. Pathogenesis ... 27 5.3. Prevalence ... 28 5.3.1. Predisposition ... 28 5.3.2. Primary causes ... 29 5.3.3. Secondary causes ... 29 5.3.4. Perpetuation ... 30
5.4. Bacterial and fungal agents ... 31
5.5. Clinical manifestation ... 32
5.6. Diagnosis ... 32
5.7. Treatment ... 33
6. ANTIBIOTIC USAGE ... 35
6.1. Antibiotics throughout history ... 35
6.2. Current global scenario ... 36
6.2.1. Zoonotic potential ... 36
6.2.2. Resistance in the veterinary scenario ... 36
6.2.3. Resistance with regard to otitis ... 38
III. L-MESITRAN® IN THE MANAGEMENT OF CANINE OTITIS EXTERNA – A PILOT STUDY ... 40
1. OBJECTIVES OF THE STUDY ... 40
2. MATERIALS AND METHODS ... 40
2.1. Study Design ... 40
2.2. Participants ... 41
2.3. Treatments ... 42
2.3.1. Treatment presentation ... 42
2.4. Phase I: Tolerance study ... 42
2.4.1. Comfort assessment ... 42
2.4.2. Glycemia assessment ... 43
2.5. Phase II: Clinical Study ... 43
2.5.1. Schedule ... 43
2.5.2. Clinical examination ... 44
2.5.3. Cytological examination ... 45
2.5.4. Antimicrobial culture, susceptibility testing & biocidal activity testing ... 46
2.5.5. Sample Size ... 46
2.5.6. Efficacy analysis and outcome measurements ... 46
2.5.7. Owner feedback ... 46
2.5.8. Withdrawal & Clinical failure ... 47
2.5.9. Statistical Analysis ... 47
3. RESULTS ... 47
3.1. Phase I Results ... 47
3.1.1. Comfort assessment ... 47
3.1.2. Glycemia assessment ... 48
3.2. Phase II Results ... 48
3.2.1. Animals included in the study ... 48
3.2.3. Statistical Analysis ... 50
3.2.4. Cytological progression ... 52
3.2.5. Antimicrobial culture, susceptibility testing and biocidal activity testing ... 53
3.2.6. Owner feedback ... 54
3.2.7. Follow-‐up ... 54
4. DISCUSSION ... 55
4.1. Overall evaluation of L-‐Mesitran® in the treatment of otitis externa ... 55
4.1.1. Clinical and cytological progression ... 55
4.1.2. Significance of cytological results ... 56
4.1.3. Significance of microbiological results ... 56
4.2. Treatment formulation, administration & owner compliance... 57
4.3. Weaknesses of this trial ... 58
4.4. Other considerations ... 59 5. CONCLUSION ... 60 6. FUTURE PROSPECTS ... 61 BIBLIOGRAPHY ... 63 ANNEX I ... 73 ANNEX II ... 74 ANNEX III ... 75 ANNEX IV ... 76 ANNEX V ... 77
LIST OF FIGURES
Fig. 1 - Post-surgical infection. ... 24
Fig. 2 - Post-antibiotic treatment. ... 24
Fig. 3 - Start of L-Mesitran®. ... 24
Fig. 4 - 1 day after L-Mesitran®. ... 24
Fig. 5 - Healed at 3 weeks. ... 24
Fig. 6 - Start of L-Mesitran®. ... 25
Fig. 7 - 2 weeks after L-Mesitran®. ... 25
Fig. 8 - Healed at 1 month. ... 25
Fig. 9 - Post-amputation & start of L-Mesitran®. ... 26
Fig. 10 - After 1 month of treatment. ... 26
Fig. 11 - Fully healed at 6 weeks. ... 26
Fig. 12 - Representation of the canine auricular canal (original source). ... 31
Fig. 14 - Example of 21-day treatment schedule per enrolled canine in the clinical trial. .. 44
Fig. 15 - Survival analysis of probability of clinical cure over time ... 50
Fig. 16 - Survival analysis of probability of clinical and cytological cure over time ... 51
Fig. 17 - Box plot evaluating clinical score decrease. ... 51
Fig. 18 - Box plot evaluating owner VAS score decrease. ... 52
Fig. 19 - Malassezia sp. - Day 0 (x400 amplif.). ... 52
Fig. 20 - Improvement – Day 21 (x400 amplif.). ... 52
Fig. 21 - Rods & cocci-Day 0 (x1000 amplif.). ... 53
Fig. 22 - Improvement-Day 21 (x1000 amplif.). ... 53
LIST OF TABLES
Table 1 – Common predisposing factors of otitis externa ... 29
Table 2 – Common primary causes of otitis externa ... 29
Table 3 – Common secondary causes of otitis externa ... 30
Table 4 – Common perpetuating factors of otitis externa ... 30
Table 5 – Inclusion criterion for dogs ... 41
Table 6 – Exclusion criterion for dogs ... 41
Table 7 – Clinical signs and respective scores (according to Nuttal & Bensignor, 2014). . 45
Table 8 – Weekly clinical scores per ear for 26 ears ... 49
LIST OF ABBREVIATIONS
CAD Canine Atopic Dermatitis
CADESI-04 Canine Atopic Dermatitis Extent and Severity Index-04
CE European Conformity
CFU Colony-forming Units
ESBL Extended-spectrum Beta-lactamase FDA Federal Drug Administration H₂O₂ Hydrogen peroxide
IL Interleukin
LAB Lactic acid bacteria LL/2 Lewis Lung Carcinoma/2
MBC Minimum Bactericidal Concentration MIC Minimum Inhibitory Concentration
MM6 MonoMac-6
MRSA Methicillin-resistant Staphylococcus aureus
MRSP Methicillin-resistant Staphylococcus pseudintermedius OTIS3 0-3 Otitis Index Score
PAI Plasminogen activator inhibitor PBS Phosphate buffered saline ROS Reactive oxygen species SPF Specific-pathogen-free TNF-α Tumor necrosis factor-α VAS Visual analog scale
I. INTRODUCTION
1. REVIEW OF CURRICULAR INTERNSHIP – WEST CROSS VETERINARY HOSPITAL, JAPAN
The initial half of the mandatory curricular internship took place at West Cross Veterinary Hospital in Tokyo, Japan, under the supervision of owner and head veterinarian, Dr. Nobuyori Tsukagoshi. This location was chosen for various reasons ranging from the curiosity of how veterinary practice differs on the other side of the world to the desire of experiencing living as a local resident in Tokyo. Seeing as language was going to be an enormous and possibly impeditive barrier, West Cross Veterinary Hospital was the appropriate choice due to it´s bilingual environment and Dr. Tsukagoshi´s veterinary background at an American university. While the use of Japanese language was a pre-requisite for interning there, fluency was not. English was used constantly and required when attending to foreign owners who did not speak Japanese either. West Cross´s location was also highly appealing as it tended to the needs of residential areas known for its many foreigners and a community of people who cared dearly about and were able to invest in treatment for their pets.
During over 500 hours I was able to participate actively in the clinic´s activities, which ranged from general clinical practice to general surgery. When consultations were in Japanese I observed firstly and paid attention to the anamnesis and any questions I had regarding the case were addressed in private to Dr. Tsukagoshi. If the owners happened to be from a country other than Japan I would begin the consultation and then communicate the information to my supervisor. This interaction with the foreign owners was a very positive experience in the sense that I was given more responsibility towards them and the bond was stronger due to the common language. With regard to practical activities in this sense I was able to review how to perform basic auxiliary diagnostic procedures such as Diff-Quick staining for cytology and use of technical equipment for biochemistry analysis and urine and blood processing.
One of the most important aspects of the practical component at West Cross was the large emphasis on ultrasound diagnostic skills. The clinic had the convenience of possessing its own ultrasound machine for immediate diagnostic aid during the consultation itself and this allowed for much learning and hands-on experience. Basic normal anatomy was the first area that we focused on and from there we approached pathological situations. This constant practicing was extremely useful, as I personally felt that diagnostic imaging had always been one of the most challenging to me. Towards the end of my internship I was encouraged to
make my own diagnosis and transmit it to the rest of the staff, after which one of the attending veterinarians would confirm it. There were various scenarios in which I was instructed to communicate the diagnosis to the owners and perform the ultrasound exam in their presence whilst explaining to them what appeared on the screen during the exam. Once I had become more accustomed with medical and anatomical terminology in Japanese I began to do the same with the local pet owners. The same kind of exercise was applied to echocardiography, though with slightly less frequency.
2. REVIEW OF CURRICULAR INTERNSHIP – FACULTY OF VETERINARY MEDICINE, UNIVERSITY OF LISBON, PORTUGAL
The second half of the internship focused entirely on the area of clinical dermatology and immuno-allergology, under the close mentoring of Professor Ana Mafalda Martins, at the FMV teaching hospital. During this time I accompanied the specialty consultations and after a short period of observation commenced with obtaining patients´ anamnesis. This process allowed for an understanding of background context in order to develop a comprehensive rationale of the cases at hand but also provided an invaluable opportunity to exercise clinician to owner communication, a component that is crucial to clinical practice but oftentimes overlooked.
With regard to practical procedures I was taught all of the steps necessary to conduct a complete dermatological evaluation. Skin scrapings, both superficial and deep, were made in order to assess for presence of mites and combing of the hair coat for fleas or ticks, for example. Evaluation of the hair condition was done through hair plucking and such information provided evidence ranging from self-inflicted trauma to infection to parasitic infection to genetic conditions of the hair coat.
Another interesting aspect of the internship in dermatology and immuno-allergology was the observation of allergic reactions through intradermal skin tests. These were also an example of dermatology´s many diagnostic procedures which allow for immediate conclusions and answers.
One of the most valuable assets that I will take from this half of the internship is undoubtedly the value of microscope as a crucial tool in aid and confirmation of dermatological diagnosis. Learning to collect samples and stain them in order to be observed and interpreted was essential and many times provided the convenience of being able to make an immediate diagnosis and if not, an exclusion of one. Identifying common microorganisms from cocci to rods to yeasts and learning to interpret infection or absence of it by quantifying them helped
the process of evolution as a future clinician and could only be perfected through constant practice. On some occasions biopsy samples were required and I was taught the correct procedures for such, many times with use of a biopsy punch.
Seeing as the subject of this thesis pertains to otitis externa a large portion of my time at the hospital was concentrated on the ears and the final stages of the internship were dedicated entirely to patients with ear disease. Knowledge of basic routine otoscopy was acquired throughout consultations and the goal of visualizing intact tympanic membrane or absence of it was accomplished. Auricular cytologies were also performed and stained for viewing under the microscope and where it was deemed necessary samples were also collected for microbial culture and sent to the in-house laboratory. For cases in which the tympanic membrane could not be observed with use of a simple otoscope or in instances of doubt I was taught to make use of a video-otoscope. Such device allowed for direct and clear observation of the entire ear canal and permitted a very detailed evaluation of the internal condition of the ear. When animals presented with excessive and/or solidified cerumen, thus impeding visualization of the canal, a lavage was necessary so as to permit the examination. The video-otoscopic examinations allowed for familiarization with the ear´s anatomy, one which is quite elaborate and fragile. In cases of suspected or confirmed presence of foreign bodies within the ear, removal procedures were conducted. These instances called for careful and precise manipulation of fragile equipment in an even greater fragile environment, that of the ear. Perhaps the most rewarding aspect of this internship was the opportunity to observe patient progression and the privilege of establishing relationships with owners and their companion animals. My previous notion of clinical practice was that of numerous different patients and cases, most of which would be seen once by a clinician and then perhaps seen by another during a follow-up, if a follow-up were called for at all. The great difference between specialty consultations, in this case, dermatology, is that one can accompany the evolution and monitor firsthand the results and constantly receive owner feedback, making practice even more rewarding on a personal level. I believe that there is great value in creating trust between owners and veterinarians and that communication and listening skills are key to maintaining owner loyalty and through being present at these consultations with Professor Ana Mafalda I was able to determine what kind of a clinician I myself would like to be.
II. REVIEW OF LITERATURE 1. HONEY
1.1. Honey throughout history
“There comes forth from their bellies, a drink in varying colour wherein is healing for men” (Quran 16:69, Mohsin Khan). Before the world knew sugar, there was honey. Since pre-historic times it has been depicted as a part of human life, as numerous pieces of artwork dating from the Stone Age indicate. The earliest evidence is a cave painting of over 10000 years in eastern Spain, which shows the arduous quest by man to collect honey from a beehive and Ancient Egyptian hieroglyphs hint at the domestication of bees through reference to clay hives (McGee, 2004).
The first written reference to honey interestingly refers to its medicinal use and lies in a Sumerian tablet from 2100-2000 BC, in which there is mention of it as a drug and ointment (Mandal & Mandal, 2011). A number of Egyptian papyruses also later made reference to honey as being prescribed for external use in various conditions, post-operatory treatments, as an anti-inflammatory and even as a suppository (Bogdanov, 2014).
Though written reference to honey refers to its healing properties it is clear that throughout history many civilizations regarded it not only as an important food source but also as a symbol in religion and ceremonies. Hajar (2002) outlines some of the most interesting historical uses for honey among different civilizations. In Greek mythology the almighty Zeus avoided being eaten by his father thanks to the bee-nymph Melisseas, who fed him the honey, which made him strong enough to seize the throne. It is said that Cleopatra´s cosmetics were honey-based and other women from Arabia valued its softening properties and applied it as a facial mask. Pharaohs utilized honey in wedding celebrations, during which the newlyweds would drink honey for good luck and happiness, such that the term honeymoon originated from this time and was then passed on to Greco-Roman culture, still used to this day. Honey was a common offering to the gods in Ancient Egypt and the dead of nobility were buried in or with jars of honey. Tutankhamen´s tomb was found to enclose vast quantities of honey in jars and it is said that Alexander the Great himself was buried in honey (Hajar, 2002).
1.2. Honey production
The Codex Standard for Honey (1981) describes it as the sweet substance produced by honeybees from various plant nectars or by collecting and transforming the excretions of insects that live by sucking portions of plants. Molan (2012a) states that honey is mostly produced by the honeybees from the nectar obtained from different flowers, yet they may also collect the phloem sap of plants in the form of honeydew, which drips after activity by aphids.
Honeybees (Apis mellifera) are extremely advanced insects that live in a complex organizational structure, similar to that of a society. A bee colony consists of various members assigned to a number of different cargos entailing specific duties, all of which ultimately answer to one large queen. With tasks ranging from food collection to habitat defense to communication, bees work in a methodically orchestrated manner, much more evolved than that of solitary insects (Mid-Atlantic Apiculture Research and Extension Consortium [MAAREC], 2014). Forager bees, for example, are tasked with collecting the nectar from flowering plants by drinking it and storing it within their crop, or honey stomach, though no digestion occurs there. These such bees then take the nectar back to their hive and regurgitate it directly into the crop of a processor bee, after which they return to the flowers to repeat the cycle. The processor bees then regurgitate the nectar into hexagonal wax cells within the honeycomb for ripening (Shipman, 2013).
The ripening process is a collective one and involves enzyme secretion on behalf of the honeybees every time regurgitation occurs, particularly invertase, which promotes the breakdown of sucrose into glucose and fructose. The nectar is composed largely by sucrose and water. Next the bees remove the water content from the nectar, or dry it, by fanning their wings to create airflow around the honeycomb and aid in its evaporation (Shipman, 2013). In the end this process forms a thick syrup that remains sealed in the hexagonal cells of the honeycomb (Molan, 2012a).
1.3 General properties of honey
“Honey is a natural sweetener, but it is not just a sweetener it´s nature´s gift to mankind” (Singh et al., 2012, p. 12). Honey, in its essence, is a supersaturated viscous solution with a carbohydrate content of 80-85%, most of which is integrated by sucrose, glucose and fructose (Buba, Gigado & Shugaba, 2013; Molan, 2012a). Nearly all of the sucrose is changed into glucose and fructose, which in the end account for up to 90% of honey´s total sugar content (Molan, 2012a).
Before advances in research were made regarding the precise composition of honey it was believed that the monosaccharides glucose and fructose and the disaccharide sucrose integrated it entirely. However, with the evolution in techniques for separation and analysis of sugars, 22 other more complex sugars were found to be present in honey, although glucose and fructose account for the vast composition (Matej, 2004). Such complex sugars end up accounting for 10% of the total sugar content of honey (Molan, 2012a). Curiously, most of these sugars are not found directly in the nectar but are results of the enzymes generated by
honeybee activity, especially invertase, during the ripening of the honey or through chemical action of the acid-sugar mixture in the honey itself (Matej, 2004; White & Doner, 1980).
1.3.1. Enzymes of honey
There are numerous enzymes present in honey, all of which contribute to its functional properties, making it a unique sweetener when in comparison to others, with the most predominant being invertase, diastase and glucose oxidase (Ropa, 2013). The honeybee secretes invertase from its salivary glands and into the honey sac, where the enzyme hydrolyzes the breakdown of sucrose into glucose and fructose, in other words, inverts sugar (Matej, 2004; Ropa, 2013). This enzyme also catalyzes the synthesis of more complex carbohydrates, as it integrates a slightly reversible reaction. When invertase is present in processed or sealed honey it continues to promote the breakdown of sucrose to ripen and mature whilst in storage (Matej, 2004).
Diastase digests starch to simple compounds such as maltose and is also added to the nectar by the honeybees during the collection and ripening processes of honey (Buba et al. 2013; Ropa, 2013). This enzyme´s function is unknown seeing as no starch is present in honey but it has been used as an indicator of quality in European countries, presumably due to its varying levels in different types of honey and its ability to be measured. Despite this common practice, diastase levels do not correlate with honey quality, as its levels can be affected by numerous factors such as floral origin, bee foraging patterns, pH variations and long storage conditions with varying temperatures (Ropa, 2013).
Gluxose oxidase is secreted from the honeybees´ hypopharyngeal gland and into the nectar to aid in honey formation. It catalyzes the conversion of glucose to gluconolactone, which then yields gluconic acid, the principal acid in honey and hydrogen peroxide, which greatly accounts for honey´s antibacterial effect (Matej, 2004; Ropa, 2013). The slightly acidic pH of honey is attributed to this and to other organic acids and is responsible for differences in taste among various types of honey (Matej, 2004).
The enzyme catalase, on the other hand, works in an opposite manner to that of glucose oxidase in that it hydrolyzes hydrogen peroxide to oxygen and water (Brudzynski, Abubaker, St-Martin & Castle, 2011). While the latter enzyme is formed by bees and thus depends on the age and health of the foragers (Pernal & Currie, 2000, as cited in Brudzynski et al., 2011, p. 1) as well as the quality and nature of their diet (Alaux et al., 2010, as cited in Brudzynski et al., 2011, p. 1), the former is originated from flower pollen (Brudzynski et al., 2011; Weston, 2000). The levels of hydrogen peroxide in a given type of honey are therefore determined by its respective levels of the enzymes glucose oxidase and catalase (Weston, 2000).
1.3.2. Other components of honey
In addition to its primary composition of sugar and water, honey also contains numerous other substances such as mineral and nitrogenous compounds to vitamins and trace elements of nutrition (Eteraf-Oskouei & Najafi, 2013; Molan, 2012a). Mineral compound concentration ranges anywhere between 0.1% to 1.0%, with potassium as the major component, followed by calcium, magnesium, sodium, sulphur and phosphorus (Eteraf-Oskouei & Najafi, 2013). Nevertheless, with regard to edible honey, the quantities of such compounds are much too low to be considered of any nutritional value in relation to the recommended daily intake. Besides these elements, honey also contains diverse polyphenols, such as flavonoids, which possess significant antioxidant activity, possibly also contributing to honey´s healing properties (Molan, 2012a).
1.4. Healing properties of honey
The clinical application of honey was abandoned in modern Western medicine due to the discovery and rise of antibiotics, becoming essentially limited to traditional medicine in certain cultures. Currently, the incomplete knowledge regarding the antibacterial characteristics of honey in combination with variability among activity pose large obstacles for the return of its applicability in modern medicine (Kwakman & Zaat, 2012). “ (…) the time has now come for conventional medicine to lift the blinds off this ´traditional remedy´ and give its due recognition” (Zumla & Lulat, 1989, p. 385).
The antibacterial activity of honey was initially attributed to its high sugar content, with the consequent osmotic process thought to be responsible for disrupting bacterial cells by drawing out their water content (Molan, 2012b). In 1892, Dutch scientist Van Ketel was able to demonstrate honey´s bactericidal activity (as cited in Dustmann, 1979). In 1919 a study by Sackett would also contradict the previous belief that sugar was responsible for the major activity with a surprising result, through the observation that the antibacterial potential of honey in fact increased through the dilution of honey with water. Years later, Dold, Du & Dziao (1937) revealed the discovery of an antibacterial factor which they named “inhibine” (as cited in Molan, 2012b, p. 2). This term was utilized until 1963, when White, Subers & Schepartz showed through their studies, that inhibine was, in fact, hydrogen peroxide (H₂O₂).
1.4.1. Sugar content
Approximately 80% of honey consists of sugars, mainly glucose and fructose, with usually less than 18% water composition (Kwakman & Zaat, 2012; Molan, 2012a), such that the osmolarity is enough on its own to inhibit growth of certain bacteria and fungi (Molan, 2012b). The coupling of high sugar concentration with extremely low moisture promotes
osmotic stress that also prevents the spoilage of honey by microorganisms (Kwakman & Zaat, 2012). However, high water content that may promote excessive dilution of honey can compromise the antibacterial activity. Bacteria are much more susceptible to high sugar concentrations than are fungi, which will grow at the slightest dilution, seldom surviving in the osmolarity of honey diluted to near 10% (Molan, 2012b). Still, the most common wound-infecting specie, Staphylococcus aureus, is exceptionally tolerant of osmolarity and can survive at honey concentrations of up to 30% (Molan, 2012b). With higher dilutions the antibacterial activity of honey is no longer attributed to its sugar content and is instead promoted by other compounds (Kwakman & Zaat, 2012).
1.4.2. pH of honey
Honey has a characteristic acidic pH range of 3.2 – 4.5, which in itself is capable of being inhibitory to several bacterial pathogens, with the acidity level changing according to botanical source and geographical nature (Mandal & Mandal, 2011; Satarupta & Subha, 2014; Vallianou, Gounari, Skourtis, Panagos & Kazazis, 2014). The minimum pH values for growth of common pathogenic bacteria were obtained in a study by Mandal & Mandal (2011), which evaluated Escherichia coli (pH 4.3), Salmonella spp. (pH 4.0), Pseudomonas aeruginosa (pH 4.4) and Streptococcus pyogenes (pH 4.5).
In addition and taking into account that Staphylococcus aureus is one of the most well known pathogens in terms of global health concerns, a study analyzing the interactions between lactic acid bacteria (LAB) and S. aureus inhibition also highlighted the role of pH, among other factors (Charlier, Cretenet, Even & Le Loir, 2009). Though this study essentially encompassed food systems and the vaginal environment due to the presence of LAB and consequent fermentation and acidification, the concepts can be applied in analogous form to honey. Charlier et al. (2009) deemed a pH of 4-4.5 as likely to inhibit S. aureus, seeing as its minimum growth pH is 4.6, with optimum growth being close to neutrality. Thus, with honey´s intrinsic even more acidic pH, it is logical to assume that it will also exert the same inhibitory action upon S. aureus. In addition, seeing as information regarding Staphylococcus pseudintermedius was not found, it is probable that the same concepts also apply, due to their similarity in nature.
However, the concentration of the acid itself in common honey is low and neutralization of this acidity takes place when honey is mixed with fluid from wounds or saliva. The surrounding environment of cells contains concentrations of bicarbonate, such that the dilution of common honey by an equal volume of extracellular fluid would elevate the pH to near neutrality (pH – 6.8) (Molan, 2012b). This would essentially nullify the acidity as a
contributor to antibacterial activity in situations where significant dilution takes place (Molan, 2012b). In this sense the greater part of honey´s antibacterial activity is not owed to its pH and is instead attributed to other properties.
1.4.3. Hydrogen Peroxide
Hydrogen peroxide (H₂O₂) is considered to be the major antimicrobial factor in the majority of honeys (Brudzynski, 2011; Mohaptra, Thakur & Brar, 2011; Molan, 2012b; White et al., 1963). There is correlation between levels of endogenous hydrogen peroxide and the extent to which inhibition of bacterial growth occurs (Brudzynski, 2006; White et al., 1963). The previously mentioned study by Charlier et al. (2009) reports on such inhibition, in this case specifically of S. aureus, by means of H₂O₂. The study refers to some lactobacilli strains being able to inhibit S. aureus growth, with bacteriostatic and bactericidal effects oscillating in accordance to different concentrations (Charlier et al., 2009).
The enzyme glucose oxidase, which is secreted by the honeybees directly into the nectar during honey production becomes activated with the moderate dilution of honey, upon which it converts the breakdown of glucose into gluconic acid and hydrogen peroxide, under aerobic conditions (Kwakman & Zaat, 2012). This previous notion was demonstrated through studies conducted by White et al. (1963), which observed that activity of hydrogen peroxide took place only upon exposure to air and dilution:
Glucose Oxidase + Oxygen → Gluconic Acid + Hydrogen Peroxide
This enzymic oxidation of the glucose occurs at a very slow rate in undiluted honey and increases significantly as the honey becomes diluted. The inhibine number of any given honey, as previously mentioned, is directly related to the hydrogen peroxide concentration produced in assay plates during inhibine assay procedures, by the honey enzymes (White et al., 1963).
The presumed function of H₂O₂ is to prevent the spoilage of honey when it is in unripe state, during which the sugar concentration is not yet at levels able to prevent microbial growth. During the ripening process glucose oxidase is inactivated and regains its activity upon dilution of honey (Kwakman & Zaat, 2012). The antibacterial activity attributed to H₂O₂, therefore, is determined by a balance between its activation through glucose oxidase and its neutralization, or absence, by the addition of catalase. The peroxide activity may also be destroyed by the presence of heat (Mandal & Mandal, 2011).
H₂O₂ concentration is determined by the relative levels of glucose oxidase, which are synthesized by the bee, as well as the enzyme catalase, originating from flower pollen. The latter is an enzyme that neutralizes H₂O₂, thus destroying its activity (Mandal & Mandal, 2011). Another study assessing the mechanisms by which honey kills bacteria through the successive neutralization of individual honey bactericidal factors (Kwakman et al., 2010) also demonstrated that the addition of catalase indeed reduced hydrogen peroxide to negligible values.
There is a delicate relation between the two enzymes, as mentioned previously, which might propel the idea that since glucose oxidase is produced by the bees, which maintain the process of ripening honey in strict and narrow limits, that honeys throughout the world may not differ so much in hydrogen peroxide. However, it is imperative to remember that catalase originates from plant sources, which translate to the amount of pollen retrieved by the bees and thus will ultimately determine the final levels of hydrogen peroxide (Weston, 2000). Furthermore, seeing as glucose oxidase is sensitive to external conditions, the antibacterial activity of honey that is hydrogen peroxide-dependent will depend on its exposure to heat and light during processing and storage (Molan, 2012b).
An example of the practical application of this peroxide activity is its use in wounds, where H₂O₂ contacts with the moist environment and the enzyme glucose oxidase is activated (Creemers & Bosma, 2006). Hydrogen peroxide alone for use in wound dressings has largely gone out of use due to its inflammatory effects and the risk of cytotoxicity (Molan, 2012b). Honey has demonstrated safe and effective antimicrobial activity through the continuous supply of low levels of hydrogen peroxide over an extended period of time, in contrast to a large amount at a single time (Bang, Buntting & Molan, 2003). A study conducted on 50% solutions of honey by Bang et al. (2003) showed that hydrogen peroxide accumulated to a peak level, after which it dropped, eventually to zero after 24-48 hours. This not only supported the fact that hydrogen peroxide would not accumulate to levels considered harmful to tissues but also alerted towards the notion that the antibacterial activity attributed to this substance was limited and that such concept would have to be applied in clinical use, for example in wound dressings, which would have to be changed with appropriate frequency (Bang et al., 2003).
1.4.4. Effect on Biofilms
A study by Merckoll, Jonassen, Jeansson & Melby (2009) addressed the effects of honey on ´planktonic´ bacteria on agar plates, or bacteria tested in its most vulnerable form, versus bacteria living in biofilm, a layer of bacteria-secreted polyssacharide commonly associated
with chronic infections. The latter are protected from the patient´s immune system and the action of antibiotics, with the ability to be 1000 times more resistant to antibiotics in contrast with the more vulnerable ´planktonic´ bacteria, which are used in laboratories for antibiotic sensitivity testing (Merckoll et al., 2009). The study, therefore, evaluated honey´s effects on typical real-life situations, such as recalcitrant wounds and the bacteria embedded in biofilm. In this case (Merckoll et al., 2009) the commercially available Medihoney® (Medihoney Pty Ltd., Queensland, Australia) was used, which is a mixture of gamma-irradiated honey that includes Leptospermum species, or Manuka honey. It was compared with the commercially available culinary local unmixed forest honey (Solhøy Bigård, Østfold, Norway). The bacterial strains utilized were two Gram-positive and two Gram-negative and included a methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from a pus sample from a pediatric surgery department. It was found that both honeys slowed the growth of exponential phase bacteria from a concentration of as low as 0,8% (Merckoll et al., 2009). In terms of planktonic bacteria honey inhibited its growth even at very low concentrations and although the biofilm appeared to offer protection for the bacteria, the substances in honey were able to diffuse through the matrix, though higher concentrations were necessary. Though the Norwegian forest honey was not as effective as Medihoney® it still proved to be bactericidal. Nevertheless, culinary honeys should not be used in treating wounds since they are not sterile, as will be explained in further detail ahead.
Another study by Ansari et al. (2013) assessed honey´s in vitro effect on fungal biofilms, utilizing the common pathogen Candida albicans. The honey used for this experiment was jujube honey due to its known ability to decrease and disrupt mature biofilms. Results were obtained through scanning electron microscopy and atomic force microscopy, which indicated cellular morphological alterations on the structure of C. albicans, as well as a decrease in its biofilm thickness (Ansari et al., 2013). Such findings serve as illustration of the broad antimicrobial spectrum associated with honey in that it can also be considered an effective anti-fungal agent.
1.4.5. Effect on Colonization
As elucidated by Wolcott et al. (2010), though bacterial colonization of a wound is not necessarily considered as being detrimental to the wound healing process per se, it may lead to chronic infection if the bacteria persistently utilize the host´s defenses to the point of exhausting their immune system´s protective capacities (as cited in Westgate & Cutting, 2013, p. 1). Furthermore, Jørgensen et al. (2006) indicates chronic wound infections as being
responsible for considerable patient morbidity in association with decreased quality of life (as cited in Westgate & Cutting, 2013, p. 1).
Though in vitro studies regarding the antibacterial effects of honey exist in large numbers there are few in which honey´s activity is assessed on healthy subjects and tissues. Kwakman et al. (2008) did just this and not only studied Revamil® (Bfactory, Netherlands), a medical grade honey and its bactericidal spectrum but also observed its efficacy in reducing microbial skin colonization in healthy human volunteers. A number of microorganisms were subjected to the honey, including Escherichia coli, Pseudomonas aeruginosa, clinical isolates of Enterobacter cloacae and Klebsiella oxytoca, extended-spectrum beta-lactamase (ESBL) – producing strains of these, as well as methicillin-susceptible and methicillin-resistant strains of Staphylococcus epidermidis and S. aureus, among others. Results showed that the honey had reproducible bactericidal activity against both antibiotic-resistant and susceptible isolates (Kwakman et al., 2008).
Regarding the healthy human volunteers, this study (Kwakman et al., 2008) included an investigation to assess decrease in skin colonization on the forearm. For such, 2 patches of skin were used, over which 0,5 ml of honey was applied to one area and covered with a transparent polyurethane dressing, with the other remaining as the control. After 2 days the honey was collected from the patches and it was found that honey-treated patches showed significantly less colonization than did the corresponding untreated control patches, which instead demonstrated increased colonization (Kwakman et al., 2008).
1.4.6. Effect on angiogenesis
A study by Rossiter, Cooper, Voegeli & Lwaleed (2010) investigated the possibility of honey as an angiogeneic agent through use of in vitro analogues of angiogenesis and an endothelial proliferation assay, as well as possible cytotoxicity. In this case the types of honeys evaluated were as follows: an artificial honey solution of glucose and fructose, a common supermarket honey, Activon® (Advancis Medical Ltd, UK), which is medical grade Manuka honey and Mesitran® ointment (Triticum, Netherlands), which is also a medical grade honey product based on hypoallergenic lanolin, among other components (Rossiter et al, 2010). The results of the cytotoxicity assay revealed that only Activon® showed a significant dose-response (Rossiter et al., 2010).
With regard to the angiogenesis assay, which was based on a rat aorta ring, tubule formation was evaluated with basis on density and total length. Results for all products were similar and showed highest pro-angiogenic effect at 0,2% v/v honey. Regarding endothelial proliferation, based on multi-well plates containing endothelial cells, activity was evaluated through
photographs and analysis of the wells after staining in order to measure pseudotubule density and branching. Honey dilutions at 0,2% v/v and 0,04% v/v were found to be pro-angiogenic, while at 1% they were neutral and at 5% they were anti-angiogenic. The potential which honey has towards angiogenesis is “severely under-investigated” (Rossiter et al., 2010, p. 16) and thus this laboratory model provided a foundation for future studies.
2. ALL HONEYS ARE NOT THE SAME 2.1. Honey use in medicine
It is only in recent times that the medical profession has turned its attention once more to the ancient practice of using honey as a medicinal agent. However, many present-day practitioners are unaware that honeys vary greatly with regard to their therapeutic potential and that some are more suitable than others. Oftentimes honey is treated as a “generic medicine” among scientists and physicians, with all of the variations being overlooked (Molan, 2012c). The protocol used for honey in wound management, for example, is highly variable and depends on the clinician´s preferences, with some purchasing inexpensive honeys intended for consumption, while others opt for standardized irradiated medical honey (Carnwath, Graham, Reynolds & Pollock, 2013). Although it is fact that natural honey originating from the comb has antibacterial properties, it is not of medical grade and is thus contra-indicated for wound care. “All honeys are not the same and do not possess the same therapeutic advantages; therefore, honey should not be considered as a generic term” (George & Cutting, 2007). Further yet, the antibacterial potency among numerous honeys varies in accordance with the inhibine number, or number of dilution steps a sample of honey could be subjected to while still retaining antibacterial activity. Such variance among honeys calls for careful study prior to their selection for use in clinical treatments. Nevertheless, much of the published research, whether clinical or microbiological, has been conducted without knowledge of the actual antimicrobial activity, with the minimum inhibitory concentration (MIC) sometimes varying 100-fold between different honeys (Molan, 2012b).
A study by Cooper & Jenkins (2009) compared the antibacterial activity of 17 samples of table honey purchased from British supermarkets with medical grade Manuka honey (Manukacare® 18+, Comvita, UK). The inhibitory potential of each honey sample was estimated through determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against 6 bacterial cultures, as follows: 2 laboratory reference cultures – S. aureus NCTC 6571 and E. coli NCTC 10418, as well as 4 clinically isolated bacteria from chronic wounds – MRSA, Streptococcus pyogenes, S.
epidermidis and P. aeruginosa. The results of this study (Cooper & Jenkins, 2009) revealed antibacterial activity in 9 of the honey samples, with the medical grade honey having the greatest bactericidal action against the tested cultures.
In addition, a wide variety of microorganisms were recovered from the 18 table honeys (Cooper & Jenkins, 2009). Though most were mesophilic aerobic bacteria and were not usually considered to be pathogens, there were some capable of colonizing chronic wounds, such as Clostridium ramosum and Staphylococcus warneri. Bacillus species were the most recovered and were present in 14 samples of honey. No organisms were detected in the medical grade honey, as the sample complied with specific regulations, having been irradiated for sterilization. In summation, this study interestingly showed that not only is the antibacterial activity of common culinary honeys unreliable and sometimes non-existent, but the potential presence of pathogenic organisms in them limits their use in medicine (Cooper & Jenkins, 2009).
With regard to the specific utilization of honey in the treatment of wounds, though it may be generally assumed that even honeys with low antibacterial activity will be suitable, it is imperative to consider the factors which will alter the microenvironment and hence the potency of the honey. In open wounds fluid will seep out and dilute the honey, thus decreasing its activity and when infections are in the mouth or stomach, saliva and gastric fluid will dilute the honey and also decrease its action (Molan, 2012c).
2.2. Manuka honey
The currently available research on honey´s action pertains mainly to two principal groups which vary in respect to the component involved in antibacterial activity. These are the European and American honeys, possessing catalase-sensitive activity and correlation with internal hydrogen peroxide and the Leptospermum spp honeys, which are independent of hydrogen peroxide and are instead active with basis on an internal component named methylglyoxal (Brudzynski, Abubaker & Wang, 2012). Manuka honey is set apart from other honeys in that it is a non-peroxide honey, therefore retaining its full antibacterial activity in the presence of catalase and in contrast to other honeys (Molan, 2012g). It is produced in most abundance in New Zealand, where Manuka trees grow uncultivated over large territory but is also produced in Australia, although in much lesser quantities, from Leptospermum scoparium trees (Molan, 2012g).
Though Maori tribes used manuka honey as a medicinal agent, its principal antibacterial component, methylglyoxal, is not integrated in the nectar collected by the bees to make honey and is instead formed by a chemical reaction occurring after the bees have processed the
nectar into the honey (Molan, 2012g). Manuka honey is currently being sold with four different rating scales of antibacterial activity and even when the same scale is used, variations can result due to different laboratory procedures, for example. (Molan, 2012g). 2.3. Medical grade honey
The term ´medical grade honey´ is largely used as a marketing term and there is no official definition as to what it is or what it must be. True medical grade honey must comply with a number of strict criteria and differs greatly from common table honeys, as previously mentioned. As elaborated by Postmes, van den Bogaard & Hazen,
Like other natural products, the composition of honey is not constant, and, moreover, it may contain residues of pesticides or drugs such as tetracyclines that are used for treatment of bee diseases. In most countries honey for human consumption must be checked for residues; however, for medical use higher quality standards are needed. It seems advisable to use only honey derived from specific-pathogen-free (SPF) hives, which have not been treated with drugs, and are gathered in areas where no pesticides are used. Honey intended for medical use should be sterile and free of residues, which might make the clinical use of honey more acceptable (The Lancet, 1993, p. 756).
Regardless of the existence or absence of an official definition for what medical grade honey is, it is imperative that it be subjected to measures which will guarantee its safe clinical use, one of which is the sterilization procedure through gamma irradiation (Postmes et al., 1993; Molan & Allen, 1996). Furthermore the various types of honeys produced under this category undergo, in addition to the sterilization, careful filtration and are produced under exacting standards of hygiene (George & Cutting, 2007). In recent years numerous medical grade honey products composed of different types of honeys at different concentrations have been approved by the European Union for use in wound care and are being employed successfully (Stobberingh & Vandersanden, 2010).
2.3.1. Variation among medical grade honeys
Despite proven efficacy of the numerous commercially available medical grade honey products and their benefits over common table honeys, there are differences among the former regarding antibacterial activity. Stobberingh & Vandersanden (2010) set out to compare the in vitro activity of commercially available products against clinical isolates containing antibiotic resistant strains of S. aureus and P. aeruginosa. Among the products evaluated were pure honey formula Revamil® (B-factory, NL), pure Manuka honey (Activon, UK), L-Mesitran® Soft, containing 40% honey (Triticum, NL) and L-Mesitran® Ointment, containing 48%
honey (Triticum, NL). Results for the study (Stobberingh & Vandersanden, 2010) revealed that Revamil®, for example, was bactericidal against P. aeruginosa but was not effective against S. aureus. Moreover it was demonstrated that L-Mesitran® Soft was by far the most effective of the products in terms of antibacterial activity and required the least amount of product to obtain such efficacy, meaning that potential was observed at the lowest concentration in comparison to others. The other products would need relatively more material in order to reach the same level of antibacterial activity, with the exception of Revamil®, which showed no significant activity against S. aureus (Stobberingh & Vandersanden, 2010).
2.3.2. Risks & gamma irradiation
The risk most notoriously associated with honey use is that of botulism due to the presence of clostridial spores and gamma irradiation has been found to kill any such spores, allowing for a sterile product without loss of antibacterial activity (Merckoll et al., 2009). Furthermore, Creemers & Bosma (2006) advise against the use of common retail honey in treating wounds, as it is probable that these may contain traces of pesticides and herbicides which may pose a risk of toxicity. In addition to pesticides and herbicides, Stobberingh & Vandersanden (2010) also mention other dangers such as heavy metals and antibiotics used to treat diseases in bees. Seeing as it has been well established that honey´s antibacterial activity is heat labile, sterilization through autoclave would not be viable and would thus destroy its beneficial characteristics. Postmes et al. (1993) utilized gamma irradiation on 2 batches of lime honey, thus contributing to the viability of honey use in a clinical context. One batch contained 520 colony-forming units (CFU) per 100 grams of honey, with 40 CFU identified as Clostridium perfringens and the rest Bacillus spp, with the other batch containing 4200 CFU Bacillus spores per 100 grams. Irradiation with 18 kGY successfully sterilized both batches without compromising their antibacterial activity (Postmes et al., 1993). Molan & Allen (1996) also investigated the effect of gamma irradiation (25 kGy) on the same antibacterial activity of honey and found that there were no significant changes in such. In the aforementioned study (Molan & Allen, 1996) 5 honeys were utilized, with 2 having their activity attributed to hydrogen peroxide and the other 3 being manuka honeys with non-peroxide activity. The honeys were tested against S. aureus in an agar well diffusion assay and even when doubling the radiation to 50 kGy antibacterial activity was maintained. Nevertheless, testing of honey containing spores of Clostridium perfringens and Clostridium tetani indicated that sterility was achieved at 25 kGy (Molan & Allen, 1996.)
In essence, true medical grade honey must be able to guarantee its clinical safety through the elimination of potential pathogenic organisms by gamma irradiation, without compromise of and whilst preserving its full antimicrobial and healing properties.
2.4. Honey as a medical device
The use of honey, as previously mentioned, has been recorded throughout history and it appears to have been a standard form of treating wounds until the appearance of antibiotics in the 1940s. It has even been said by doctors in some reports, that the idea of re-using honey was provided by older nursing staff, who recalled it being used in the past (Molan, 2012d). As more research arises regarding the antimicrobial properties of honey, an alternative medicine branch named apitherapy has been developed, which offers treatments based on honey and other bee derivatives against many conditions, including bacterial infections (Mandal & Mandal, 2011).
Following the increasing publications and reports in medical journals regarding honey´s favorable results in the clinical environment, particularly in wound dressings, two developments resulted according to Molan (2012d): practitioners adhered to the use of honey in wounds and companies started to produce sterilized honey wound dressings as registered medical devices. The European Commission´s Directorate General for Enterprise states that,
Medical devices are defined as articles which are intended to be used for a medical purpose. The medical purpose is assigned to a product by the manufacturer. The manufacturer determines through the label, the instruction for use and the promotional material related to a given device its specific purpose. As the directive aims essentially at the protection of patients and users, the medical purpose relates in general to finished products regardless of whether they are intended to be used alone or in combination (Medical Devices: Guidance document, 1994, p.3).
Within the vast field of products that can be considered medical devices, medical grade honey products are under the category for non-invasive devices, which are further classified in accordance to specific European Commission´s Directorate General for Health and Consumer. In general the medical honey products follow rule number 4 of the Commission, which addresses devices “In contact with injured skin (mechanical barrier, compression, absorb exudates)” (European Commission´s DG Health and Consumer, 2010, p.17), which then leads to a final classification of IIb, that according to the Commission is “Intended for wounds which breach dermis and heal only by secondary intent” (European Commission´s DG Health and Consumer, 2010, p.17).
2.5. Generalities in wound healing
Molan (2012e) simplifies the wound healing process so as to understand how honey works in promoting it. The rate-limiting factor in healing is oxygen supply from newly formed blood capillaries, since oxygen does not dissolve in at a fast enough speed from the small surface area of the wound. New cells can only grow in a moist environment, such that if a wound dries out the surface is covered by a scab formed from dried wound fluid and new tissue can only grow beneath this. The result is a delayed overall repair process and a scarred surface where the scab used to be since no repair took place in the dry area (Molan, 2012e). Honey promotes granulation, epithelialization, as well as reduces the amounts of exudate (Al-Waili, Salom & Al-Ghamdi, 2011) and keeps bacteria out of the wound, as will be discussed.
Supporting results are also found in another study using honey for the topical treatment of skin wounds in mice (Ghaderi & Afshar, 2004). Formation of granulation tissue and activation of fibroblasts was increased by honey, in addition to greater thickness of the basement membrane and epidermis, as well as of the collagen fibers. In comparison with the control group, which received a simple dressing with sterile gauze, the honey-treated group demonstrated constant advantage in terms of absence of inflammation, edema and dehiscence. Final conclusions of this study were that honey can accelerate the wound healing process whilst increasing resilience, tensile strength and toughness of wounds (Ghaderi & Afshar, 2004).
2.5.1. Physical barrier
Honey´s viscosity alone serves as a physical barrier which prevents bacteria from entering and keeps the wound moist in order to potentiate healing. In addition the high sugar content promotes the formation of a liquid layer between the wound surface and the dressing, since fluid is drawn out by means of osmosis. The resulting environment not only accelerates the healing process but prevents scab formation, thus avoiding scarred surface tissue. In contrast, dry dressings adhere to the surface of wounds and are a constant hindrance to tissue renovation when changed, as the newly formed tissue is torn off the surface (Molan, 2012e). 2.5.2. Wound acidification
Molan (2012e) also mentions the beneficial acidifying action of honey on wounds, which has been found to accelerate the healing rate. There are two mechanisms stated in his work, one of which regards the release of more of the oxygen that is being carried by hemoglobin in the bloodstream, seeing as oxygen is the rate-limiting factor in new cell growth, as previously mentioned. The other involves the inactivation of the digestive enzymes in the wound, which may be responsible for the destruction of newly repaired tissues or the growth factors required
in order to stimulate new tissue formation. Such enzymes operate at a neutral pH and therefore honey´s more acidic pH inactivates their activity (Molan, 2012e).
2.5.3. Debriding action
“Debriding is the medical term for removing from the surface of a wound any attached pus and/or dead tissue” (Molan, 2012e). This process is essential to wound healing, as the remaining of pus or dead tissue on a wound will generate an ongoing inflammatory response and thus prevent permanent healing. In addition to the inflammatory response the presence of pus provides a favorable environment for bacteria to proliferate. Honey is a rapid and effective debriding agent in comparison to other pharmaceutical products claiming the same action (Molan, 2012e; Morris, 2008). The explanation for such is that honey stops white blood cells from producing plasminogen activator inhibitor (PAI) that normally prevents the activation of plasmin in wound tissue. These clots are responsible for the attachment of pus and debris to the wound surface. Since the honey stops the production of PAI, it allows for more plasmin to be activated and thus digest the fibrin clots, culminating in the removal of pus and debris (Molan, 2012e).
2.5.4. Deodorizing effect
The foul odor commonly associated with infected wounds, particularly when anaerobic bacteria are present, has been motive for discomfort among patients and honey exerts rapid effect in eliminating it (Al-Waili et al.; 2011; Molan, 2012e; Morris, 2008). The unpleasant odor is associated with the breakdown of proteins in wound tissues by bacteria, which generates sulphur compounds and amines. Honey swiftly deodorizes wounds by simply providing the bacteria with glucose as a source of alternative energy, which is preferred over protein and through which no malodorous components arise (Molan, 2012e).
2.5.5. Anti-inflammatory effect
The exact mechanisms through which honey exerts anti-inflammatory action have yet to be studied and clarified but the evidence for such is large and continuously expanding in the scientific world. A study comparing the effectiveness of Indonesian honey, Manuka honey and a control hydrocolloid dressing on the rate of wound healing in mice (Haryanto, Urai, Mukai, Suriadi, Sugama & Nakatani, 2012) revealed both honeys to have the upper hand. The mice had induced wounds which were treated with the 3 comparative substances and evaluated throughout 14 days. Macroscopic results on days 2, 5 and 7 showed smaller wound areas in both honey groups, with newly formed granulation tissue and epithelium, whereas the control group had larger wound areas. Microscopic analyses on day 3 revealed greater
